There is a growing need for analysis of biomolecules, including proteins, polypeptides and DNA. Capillary electrophoresis (CE) is a process for separating molecules based on their size or charge. In capillary electrophoresis molecules are introduced into a fluid-filled capillary tube and subjected to an electric field (see, Kemp, G. (1998) “CAPILLARY ELECTROPHORESIS: A VERSATILE FAMILY OF ANALYTICAL TECHNIQUES,” Biotechnol. Appl. Biochem. 27:9-17; Wu, D. et al. (1992). Capillary electrophoresis techniques are reviewed by Schwartz, H. et al. (“SEPARATION OF PROTEINS AND PEPTIDES BY CAPILLARY ELECTROPHORESIS: APPLICATION TO ANALYTICAL BIOTECHNOLOGY,” Beckman BioResearch Literature No. 727484).
Capillary electrophoresis (CE) has become an attractive alternative to traditional slab gel electrophoresis for biomolecular separations due to its ability to provide fast, highly efficient sample separations with minimal sample volume requirements (Monton, M. R. (2005) “RECENT DEVELOPMENTS IN CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY OF PROTEINS AND PEPTIDES,” Anal Sci. 21(1):5-13). Numerous approaches for accomplishing capillary electrophoresis have been previously described (see, for example, U.S. Pat. Nos.: RE37,606; 6,440,284; 6,436,646; 6,410,668; 6,372,353; 6,358,385; 6,355,709; 6,316,201; 6,306,273; 6,274,089; 6,235,175; 6,153,073; 6,129,826; 6,107,044; 6,074,542; 6,068,752; 6,042,710; 6,033,546; 6,001,232; 5,989,399; 5,976,336; 5,964,995; 5,958,694; 5,948,227; 5,916,426; 5,891,313; 5,846,395; 5,840,388; 5,777,096; 5,741,411; 5,728,282; 5,695,626; 5,665,216; 5,582,705; 5,580,016; 5,567,292; 5,552,028; 5,545,302; 5,534,123; 5,514,543; 5,503,722; 5,423,966; 5,421,980; 5,384,024; 5,374,527; 5,370,777; 5,364,520; 5,332,481; 5,310,462; 5,292,416; 5,292,372; 5,264,101; 5,259,939; 5,139,630; 5,120,413; 5,112,460; 5,015,350; 4,865,706). Two primary separation mechanisms are commonly used in CE: procedures in which separations are obtained based on differences in the molecular size of analytes, and procedures in which separation is achieved by exploiting differences in the charge density (charge/mass ratio) of analytes.
“Capillary Gel Electrophoresis” (“CGE”) is used to separate analytes based on differences in their molecular size. Typically, CGE is carried out using gel matrices of controlled pore sizes. Separations result from differences in the abilities of different sized molecule to penetrate the gel matrix. Size separation is achieved because small molecules move more rapidly through the separation gel than large molecules. In order to employ CGE with polypeptides and proteins, it is generally necessary to denature the molecules (for example, with sodium dodecyl sulfate (SDS)), so that all of the analytes will have the same effective charge density. CGE is discussed in Bean, S. R. et al. (1999) (“SODIUM DODECYL SULFATE CAPILLARY ELECTROPHORESIS OF WHEAT PROTEINS. I. UNCOATED CAPILLARIES,” J. Agric. Food Chem 47(10):4246-55); Wu, D. et al. (1992) (“SODIUM DODECYL SULFATE-CAPILLARY GEL ELECTROPHORESIS OF PROTEINS USING NON-CROSS-LINKED POLYACRYLAMIDE,” J. Chromatogr. 608:349-356); Lausch, R. et al. (1993) (“RAPID CAPILLARY GEL ELECTROPHORESIS OF PROTEINS,” J. Chromatogr. 654:190-195); Manabe, T. et al. (1998) (“SIZE SEPARATION OF SODIUM DODECYL SULFATE COMPLEXES OF HUMAN PLASMA PROTEINS BY CAPILLARY ELECTROPHORESIS EMPLOYING LINEAR POLYACRYLAMIDE AS A SIEVING POLYMER.” Electrophoresis 19:2308-2316); and Ganzier, K. et al. (1992) (“High-Performance Capillary Electrophoresis of SDS-Protein Complexes Using UV-Transparent Polymer Networks,” Anal. Chem. 64:2665-2671).
In contrast, “Capillary Zone Electrophoresis” (“CZE,” also known as free-solution CE (FSCE)) separates analytes based on differences in their charge densities. These differences cause differing electrophoretic mobilities, and hence differing velocities of migration. In general terms, CZE involves introducing a sample into a capillary tube and applying an electric field to the tube. The electric field pulls the sample through the tube and separates it into its constituent parts (i.e., each of the sample constituents has its own electrophoretic mobility; those having greater mobility travel through the capillary faster than those with slower mobility). As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones. The detector measures the absorbance of light by each constituent at a specified wavelength; different constituents absorb light differently, and, because of this, the constituents can be differentiated from each other.
Two general categories of CZE can be described, depending upon the contents of the capillary columns. In “gel” CZE, the capillary tube is filled with a suitable gel, e.g., polyacrylamide gel. Separation of the constituents in the sample is predicated in part by the size and charge of the constituents traveling through the gel matrix. In “open” CZE, the capillary tube is filled with an electrically conductive buffer solution. Upon ionization of the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution (the buffer solution and the sample being analyzed), must also flow in this direction to maintain electroneutrality. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. Fused silica is principally utilized as the material for the capillary tube because it can withstand the relatively high voltage used in CZE, and because the inner walls of a fused silica capillary ionize to create the negative charge which causes the desired electroendosomatic flow (see, e.g., WO9310258A1).
To achieve optimal separation using CZE, it is important that the employed buffer solution be homogeneous and that a constant field strength be used throughout the length of the capillary. The separation relies principally on the pH controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute. Thus, the ability of CZE to separate analytes and the degree or extent of such separation can be enhanced by altering the pH of the buffer system, or by altering its ionic strength. Typically, the pH of the buffers utilized in open CZE is chosen with reference to the isoelectric points (pI) of the constituents in the sample.
CZE is discussed by Quirino, J. P. et al. (2001) (“SAMPLE STACKING OF CATIONIC AND ANIONIC ANALYTES IN CAPILLARY ELECTROPHORESIS,” J Chromatogr A. 902(1):119-135); Kasicka, V. (2004) (“RECENT ADVANCES IN CAPILLARY ELECTROPHORESIS AND CAPILLARY ELECTROCHROMATOGRAPHY OF PEPTIDES,” Electrophoresis 24(22-23):4013-4046); Kasicka, V. (2001) (“RECENT ADVANCES IN CAPILLARY ELECTROPHORESIS OF PEPTIDES,” Electrophoresis 22(19):4139-4162); Bossuyt, X. (2003) (“SEPARATION OF SERUM PROTEINS BY AUTOMATED CAPILLARY ZONE ELECTROPHORESIS,” Clin Chem Lab Med. 41(6):762-772); and Monton, M. R. (2005) (“RECENT DEVELOPMENTS IN CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY OF PROTEINS AND PEPTIDES,” Anal Sci. 21(1):5-13), in U.S. Patent Publications Nos. 2002/0029968 (Tan et al.); 2002/0055184 (Naylor et al.); 2002/0119482 (Nelson et al.); 2003/0057092 (Chien et al.); 2003/0217923 (Harrison et al.); 2003/0224436 (Nelson et al.); U.S. Pat. No. 4,483,773 (Yang); U.S. Pat. No. 4,793,920 (Cortes et al.); U.S. Pat. No. 5,120,413 (Chen et al.); U.S. Pat. No. 5,139,630 (Chen); U.S. Pat. No. 5,145,567 (Hseih et al.); U.S. Pat. No. 5,164,055 (Dubrow); U.S. Pat. No. 5,202,006 (Chen); U.S. Pat. No. 5,264,095 (Hseih et al.); U.S. Pat. No. 5,310,462 (Chen); U.S. Pat. No. 5,340,452 (Brenner et al.); U.S. Pat. No. 5,348,658 (Fuchs et al.); U.S. Pat. No. 5,405,782 (Kohn et al.); U.S. Pat. No. 5,423,966 (Wiktorowicz); U.S. Pat. No. 5,453,382 (Novotny et al.); U.S. Pat. No. 5,571,680 (Chen); U.S. Pat. No. 5,593,559 (Wiktorowicz); U.S. Pat. No. 5,599,433 (Keo et al.); U.S. Pat. No. 5,753,094 (Alter et al.); U.S. Pat. No. 5,766,435 (Liao et al.); U.S. Pat. No. 5,770,029 (Nelson et al.); U.S. Pat. No. 5,999,681 (Grabbe et al.); U.S. Pat. No. 6,007,690 (Nelson et al.); U.S. Pat. No. 6,074,541 (Srinivasan et al.); U.S. Pat. No. 6,074,827 (Nelson et al.); U.S. Pat. No. 6,344,326 (Nelson et al.); U.S. Pat. No. 6,416,642 (Alajoki et al.); U.S. Pat. No. 6,428,666 (Singh et al.); U.S. Pat. No. 6,432,290 (Harrison et al.); U.S. Pat. No. 6,475,362 (Gorfinkel et al.); U.S. Pat. No. 6,475,363 (Ramsey); U.S. Pat. No. 6,613,525 (Nelson et al.); U.S. Pat. No. 6,664,104 (Pourahmadi et al.); U.S. Pat. No. 6,686,035 (Jiang et al.); U.S. Pat. No. 6,695,009 (Chien et al.); U.S. Pat. No. 6,759,126 (Malik et al.); U.S. Pat. No. 6,764,817 (Schneider); U.S. Pat. No. 6,770,201 (Shepodd et al.); and U.S. Pat. No. 6,787,016 (Tan et al.); in European Patent Documents No. EP 0852007A1; EP 0572604A1; EP0518475A1; and EP0517370A1; and in PCT Publication No. WO9310258A1.
The high peak capacity (i.e., the number of peaks separated per unit time) of CZE makes it a desirable approach to the analysis of a wide range of biomolecules, including proteins and peptides (Kasicka, V. (2004) “RECENT ADVANCES IN CAPILLARY ELECTROPHORESIS AND CAPILLARY ELECTROCHROMATOGRAPHY OF PEPTIDES,” Electrophoresis 24(22-23):4013-4046; Kasicka, V. (2001) “RECENT ADVANCES IN CAPILLARY ELECTROPHORESIS OF PEPTIDES,” Electrophoresis 22(19):4139-4162; Bossuyt, X. (2003) “SEPARATION OF SERUM PROTEINS BY AUTOMATED CAPILLARY ZONE ELECTROPHORESIS,” Clin Chem Lab Med. 41(6):762-772); Monton, M. R. (2005) “RECENT DEVELOPMENTS IN CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY OF PROTEINS AND PEPTIDES,” Anal Sci. 21(1):5-13); nucleic acid molecules (Mitchelson, K. R. (2001) “THE APPLICATION OF CAPILLARY ELECTROPHORESIS FOR DNA POLYMORPHISM ANALYSIS,” Methods Mol Biol. 162:3-26); drugs (Hilhorst, M. J. et al. (2001) “CAPILLARY ELECTROKINETIC SEPARATION TECHNIQUES FOR PROFILING OF DRUGS AND RELATED PRODUCTS,” Electrophoresis 22(12):2542-2564), agricultural compounds (Menzinger, F. et al. (2000) “ANALYSIS OF AGROCHEMICALS BY CAPILLARY ELECTROPHORESIS,” J Chromatogr A. 891(1):45-67), and even bacteria and viruses (Kremser, L. et al. (2004) “CAPILLARY ELECTROPHORESIS OF BIOLOGICAL PARTICLES: VIRUSES, BACTERIA, AND EUKARYOTIC CELLS,” Electrophoresis 25(14):2282-2291).
Although CZE has multiple advantages, the CZE detection limit based on concentrations is far less than that of HPLC, and is not sufficient for many practical applications. The limitations of CZE reflect the very short in-capillary path length (i.e., detector window) of the flow cell of capillary tubes (typically only 1% of the path length of an HPLC flow cell). The short path length means that higher concentrations of analytes must be present in order to be detected (Shihabi, Z. K. (2000) “STACKING IN CAPILLARY ZONE ELECTROPHORESIS,” J Chromatogr A. 902(1):107-117)
In certain situations, the concentrations of analytes found in a sample may therefore be too low to permit the use of CZE separation methods. Although such samples may be concentrated using conventional methods, the resulting small volumes encumber sample manipulation, and such handling may cause a loss of analyte. In some cases the ionic profile of samples may be compromised by electrokinetic injection, leading to poor accuracy. High salt content in the sample may also lead to problems with high localized currents causing unwanted heating.
One approach to the problem of improving the sensitivity of CZE involves adjusting the capillary detection window (Quirino, J. P. et al. (2001) “SAMPLE STACKING OF CATIONIC AND ANIONIC ANALYTES IN CAPILLARY ELECTROPHORESIS,” J Chromatogr A. 902(1):119-135). Such adjustments can provide a ten-fold improvement in response. Enhanced detection means have also been employed to address the problem of analyzing dilute samples. Such means have included mass spectrometry, optical fluorescence, electrochemical oxidation or reduction, plasma resonance, radioactivity, refractive index, and conductivity. Very dilute analytes can remain undetectable despite the use of the most sensitive of known detection methods (see, e.g., Naylor et al. (U.S. Pat. No. 5,800,692)).
Another way to improve detection of dilute analytes is to concentrate the analytes prior to, or concurrent with, separation. Preseparation or concurrent analyte concentration methods, coupled with the use of a sensitive detection method, greatly increase the usefulness and efficacy of CE. Present off-line preseparation concentration methods are, however, time-consuming and suffer from various sample-handling risks such as contamination or sample loss due to spill or adsorption onto container walls. Various on-line focusing methods have been developed in response to these problems. One approach to the problem involves manipulating the composition of the sample and background solutions to cause the analyte molecules to “stack.” Stacking is obtained when ionized analyte molecules, placed in a low conductivity region of the column are induced by an electric field to move to a high conductivity region of the column. Because the low conductivity region will experience a higher electric field than the high conductivity region, analyte molecules in the low conductivity region will migrate rapidly to the barrier between the two regions, thereby causing a 10- to more than 1,000-fold enhancement in the sensitivity of detection (Quirino, J. P. et al. (2001) “SAMPLE STACKING OF CATIONIC AND ANIONIC ANALYTES IN CAPILLARY ELECTROPHORESIS,” J Chromatogr A. 902(1):119-135; Shihabi, Z. K. (2000) “STACKING IN CAPILLARY ZONE ELECTROPHORESIS,” J Chromatogr A. 902(1):107-117; Gebauer, P. et al. (2003) “THEORY OF SYSTEM ZONES IN CAPILLARY ZONE ELECTROPHORESIS,” Electrophoresis 23(12):1779-1785; Beckers, J. L. et al. (2000) “SAMPLE STACKING IN CAPILLARY ZONE ELECTROPHORESIS: PRINCIPLES, ADVANTAGES AND LIMITATIONS,” Electrophoresis 21(14):2747-2767; Beckers, J. L. et al. (2001) “SYSTEM ZONES IN CAPILLARY ZONE ELECTROPHORESIS,” Electrophoresis Oct; 22(17):3648-3658).
While such stacking is thus of some benefit, it has certain significant limitations. Significantly, although stacking improves the ability to detect an analyte, it also increases the concentration of contaminating analyte species. Stacking is possible only in situations in which the target analyte is present at a concentration below that of the background electrolytes (Beckers, J. L. et al. (2000) “SAMPLE STACKING IN CAPILLARY ZONE ELECTROPHORESIS: PRINCIPLES, ADVANTAGES AND LIMITATIONS,” Electrophoresis 21(14):2747-2767). Moreover, the ability to resolve two analytes in CZE is directly proportional to one-half the difference in their respective migration times, and inversely proportional to the sum of the standard deviations of the analyte peaks. Thus, since the size of the analyte peaks is affected by the sample volume, the use of larger sample volumes can adversely affect peak resolution (Beckers, J. L. et al. (2000) “SAMPLE STACKING IN CAPILLARY ZONE ELECTROPHORESIS: PRINCIPLES, ADVANTAGES AND LIMITATIONS,” Electrophoresis 21(14):2747-2767). Methods of accomplishing stacking are discussed by Shihabi, Z. K. (2000) (“STACKING IN CAPILLARY ZONE ELECTROPHORESIS,” J Chromatogr A. 902(1):107-117). The art has therefore sought alternative solutions to enhance the sensitivity of CZE.
Various mechanical measures have been used to facilitate the concentration of analytes. Guzman (U.S. Pat. No. 5,202,010) discloses an analyte concentrator comprising a tubular structure containing fluid-permeable end plates and a plurality of small bodies coated with antibodies or other chemical entities selected for their ability to bind to target analytes in the sample being analyzed. In operation, after being permitted to contact the sample analytes, the capillary is washed to remove excess material, and the trapped target analytes, which have been concentrated onto the small bodies of the structure, are then removed and processed for study. As will be appreciated, significant handling of the analytes is required. Guzman (U.S. Pat. No. 6,406,604) discloses an analyte concentrator having greater efficiency. The disclosed apparatus comprises a relatively large-bore transport capillary that intersects with a plurality of small-bore separation capillaries. Analyte present in the large bore capillary become captured and accumulate at the sites of intersection between the large-bore capillary and the separation capillaries. Naylor et al. (U.S. Pat. No. 5,800,692) describe a preseparation processor for use in capillary electrophoresis. The processor contains a sample processing material, preferably in the form of a membrane, gel or packed beads, for concentrating or chemically processing a sample, or catalyzing a chemical reaction. It is stated to be particularly suited to the concentration of dilute samples or the purification of contaminated samples. Zare et al. (U.S. Pat. No. 6,136,187) disclose a frit-less capillary separation device in which particles are embedded in a porous silane sol-gel matrix. Charged and uncharged molecules are embedded into the sol-gel matrix. The volatile components are allowed to evaporate, producing a hard porous glass. Different functionalized or derivatized sol-gel precursors can be used to prepare sol-gel glasses with different physical properties, such as pore size and surface charge. The porosity of the glass allows diffusion of protons and other neutral or ionic species, but restricts significant amounts of chromatographic particles from leaving the glass matrix. While the approach of Zare et al. (U.S. Pat. No. 6,136,187) provides certain advantages, considerable time is required to prepare the columns, and the requirement that the matrix be inoculated with sample prior to solidification limits peak resolution.
Thus, despite all such prior advances, a need remains for methods and apparatus that could overcome the problems of analyzing dilute samples and thereby extend the utility of CZE to permit the analysis of low concentration samples. The present invention is directed to this and other needs.